HPLC of Peptides and Proteins: Preparation and System Set‐Up

Reinhard I. Boysen1, Milton T.W. Hearn1

1 Monash University, Victoria, Australia
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.12
DOI:  10.1002/0471142727.mb1012s54
Online Posting Date:  May, 2001
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The eight basic modes of HPLC currently in use for peptide and protein analysis and purification are presented in this overview. They include: size‐exclusion chromatography (HP‐SEC), ion‐exchange chromatography (HP‐IEX), normal phase chromatography (HP‐NPC), hydrophobic interaction chromatography (HP‐HIC), reversed‐phase chromatography (RP‐HPLC), hydrophilic interaction chromatography (HP‐HILIC), immobilized metal ion affinity chromatography (HP‐IMAC), and biospecific/biomimetic affinity chromatography (HP‐BAC). In addition, some subsets of these chromatographic modes, e.g., mixed mode chromatography (HP‐MMC), charge transfer chromatography (HP‐CTC), or ligand‐exchange chromatography (HP‐LEC) are described. The unit includes overviews of the system requirements and planning strategies for set‐up of the systems.

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Table of Contents

  • The Properties of Peptides and Proteins and their Implications for HPLC Method Development
  • Detection of Peptides and Proteins in HPLC
  • Start‐Up Procedures
  • Preparation of the Sample
  • Preparation of the Mobile Phase
  • Setting up the HPLC Instrument
  • Priming of the Pumps and Low Pressure Lines with Eluents
  • Preparation of the HPLC System
  • The Gradient Delay (Dwell Volume) of the HPLC System
  • Connecting the Column
  • Programming the HPLC Instrument
  • Injecting the Sample
  • Testing the Functional HPLC System
  • Logbooks
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Campbell, I.D. and Dwek, R.A. 1984. Biological Spectroscopy pp. 255‐277. Benjamin/Cummings, Menlo Park, N.J.
   Chothia, C. 1974. Hydrophobic bonding and accessible surface area in proteins. Nature 248:338‐339.
   Dawson, R.M.C., Elliot, D.C., Elliot, W.H., and Jones, K.M. 1986. Data for Biomedical Research. Clarendon Press, Oxford.
   Dolan, J.W. and Snyder, L.R. 1989. Troubleshooting LC Systems pp. 1‐515. Humana Press, Clifton, N.J.
   Ghrist, B.F.D. and Snyder, L.R. 1988. Design of optimized high‐performance liquid chromatographic gradients for the separation of either small or large molecules. II. Background and theory. J. Chromatogr. 459:25‐41.
   Hearn, M.T.W. 1991. High‐performance liquid chromatography of peptides and proteins: General principles and basic theory. In High‐Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation (C.T. Mant and R.S. Hodges, eds.) pp. 95‐104. CRC Press, Boca Raton, Fla.
   Hearn, M.T.W. 2000. Physico‐chemical factors in polypeptide and protein purification and analysis by high‐performance liquid chromatographic techniques: Current status and future challenges. In Handbook of Bioseparation (S. Ahuja, ed.) pp. 72‐235. Academic Press, New York.
   Rickard, E.C., Strohl, M.M., and Nielsen, R.G. 1991. Correlation of electrophoretic mobilities from capillary electrophoresis with physico‐chemical properties of proteins and peptides. Anal. Biochem. 197:197‐207.
   Wilce, M.C.J., Aguilar, M.I., and Hearn, M.T.W. 1995. Physicochemical basis of amino acid hydrophobicity scales: Evaluation of four new scales of amino acid hydrophobicity coefficients derived from RP‐HPLC of peptides. Anal. Chem. 67:1210‐1219.
   Zamyatnin, A.A. 1972. Protein volume in solution. Progress Biophys. Mol. Biol. 24:107‐123.
Key References
   Dolan and Snyder, 1989. See above.
   An excellent practical manual.
   Hearn, M.T.W. 1991. HPLC of Peptides, Proteins and Polynucleotides, Fundamental Principles and Contemporary Applications. pp. 1‐776. VCH Publishers, Deerfield Beach, Fla.
   A comprehensive text encompassing most applications.
   Mant, C.T. and Hodges, R.S. 1991. High‐Performance Liquid Chromatography of Peptides and Proteins: Separation, Analysis and Conformation. pp. 1‐938. CRC Press, Boca Raton, Fla.
   A comprehensive survey of applications.
   McMaster, M.C. 1994. HPLC: A Practical User's Guide. pp. 1‐211. VCH Publishers, Inc., New York.
   A general guide to HPLC.
   Vijayalakshmi, M.A. 2000. Theory and Practice of Biochromatography. pp. 1‐721. Harwood Academic Publishers.
  A useful guide to modern chromatography.
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